With kinetic control, on the other hand, the rates of reactions depend on substrate concentrations or enzyme activities, and these enzyme activities in turn may depend on microbial growth or enzyme synthesis. The profile of products formed depends on relative rates of different competing reactions.

Biologists have focused on controlling kinetic elements of fermentation such as enzyme function, microbial activity, gene expression, or provision of substrates. However, the present analysis is based on the theory that fermentation is often controlled by thermodynamics.

—Kohn and Kim

As an example, they explain, in a mixed-culture anaerobic bioreactor, there there are several microbes that can transport a released glucose molecule into their cells and metabolize it to any number of products. The amount of energy that any particular organism can obtain depends on the concentration of all products of the reaction relative to all reactants.

Since the free glucose concentration is very low due to competition among microorganisms in the fermentation, and the products of fermentation are removed slowly, only very efficient microbes can use the low concentration of glucose at all. And they can only use it when concentrations of the products which they produce are low. Therefore, when their products start to build up, they can no longer obtain Gibbs energy by converting the reactant to a product, and they leave the glucose behind for another microorganism that produces a different product. As the different products build up, the free glucose concentration increases enabling higher concentrations of the original products. In this way, a predictable ratio of products is produced.

In chemistry, whether or not a reaction can proceed spontaneously in the forward direction is represented by the change in Gibbs energy (∆G), which can be calculated based on the ratio of products and reactants in the system. Using this calculation, a strongly negative ∆G indicates that the reaction could proceed strongly in the forward direction without the addition of energy to the system. A strongly positive value of ∆G indicates the reaction cannot proceed in the forward direction without addition of energy to the system, and it may even run in the reverse direction.

Previous research demonstrated by three different methods that the profile of products within some fermentation systems (e.g. cow’s rumen) is explained as near-equilibrium in accordance with the second law of thermodynamics. … The current studies use the theory describing the direction of metabolite flow in fermentation as a function of thermodynamics to define conditions for production of desired biofuels and for enrichment and isolation of bacteria that make those fuels. Since many fermentation systems are near equilibrium, when these systems are perturbed, the rates of reactions adjust to restore the equilibrium. In the process, microbes that carry out the favored reactions thrive and grow. Under these conditions, microbes can be enriched and isolated to produce desired products.

—Kohn and Kim

Kohn and Kim used their theory to isolate bacteria that produce a high concentration of alcohol or alkanes from cellulosic biomass or syngas (CO, CO₂, and H₂).

Cellulosic ethanol. Consolidated processing has been proposed as an efficient approach for the conversion of plant fiber directly into the fuel using a single microbial culture that digests the fiber and excretes the desired fuel as an end product. Both the digestion of the biomass and its fermentation to ethanol occur in a single step. However, the gating factor for consolidated bioprocessing is the availability of suitable microorganisms.

The microorganisms in the first stomach chamber (rumen) of a cow or other ruminant are some of the fastest microbial degraders on earth. However, ethanol is not known to accumulate in the rumen under typical conditions because the produced ethanol is converted to acetate.

Kohn and Kim hypothesized that it would be more thermodynamically favorable to make ethanol than acetate when H₂ pressure was high. Earlier work showed that when incubated in the presence of high hydrogen pressure (e.g. 1 bar H₂), mixed cultures of microorganisms from the rumen of a cow produced ethanol from glucose, 5-carbon sugars arabinose and xylose, corn starch, or cellulose.

In their experiments, they isolated microorganisms from the rumen of a cow that could convert cellulose or cellobiose directly to ethanol, or that could convert CO₂ and H₂ directly to ethanol. Rumen fluid was collected from a fistulated cow fed a grass hay diet, and fluid was enriched for fiber-digesting ethanol-producing microbes by incubating in media with timothy grass hay as the main substrate under 1 bar pressure of H₂. Every 3 to 5 days, they transferred 10% of the culture was transferred to new media and gas headspace

Microbial cultures from the rumen converted 1% cellobiose to 0.5% ethanol increasing ethanol concentration from initial 6.0% to more than 6.5% by volume. A second addition of cellobiose further increased ethanol to more than 7.0%. For different isolated strains that converted cellobiose to ethanol, 16S-rRNA sequences of strains that converted cellobiose to ethanol were >97% homologous with Clostridium (C. bifermentans, C. sordelli, C. sporogenes), Enterococcus (E. casselflavus, E. muntii, E. sangunicola, E. faecium, E. lactis), Pediococcus (e.g P. acidilactici), Lactobacillus (eg. L. mucosae), or Staphylococcus (S. epidermidis). Based on these simple isolations, it is clear that bacteria that can digest plant fiber or cellobiose to ethanol or other alcohols are common in nature and are genetically and physiologically diverse. Using reaction conditions that make alcohol production thermodynamically favorable over other potential products makes it simple to isolate such organisms.

—Kohn and Kim

Alcohols and hydrocarbons. In naerobic digestion, the degradation of acids and alkanes is favored over their synthesis (positive ∆G values when H₂ pressure is low). However, if the ratio of H₂ to CO₂ approaches 3 to 1, and if the pressure is increased, carbon dioxide fixation, and fatty acid elongation, fatty acid decarboxylation, and hydrogenation of fatty acids become thermodynamically favored. The authors hypothesized that organisms that produce higher concentration of ethanol than currently known could be isolated by incubating under such conditions that thermodynamically favor higher concentrations.

Further, they noted, higher pressures and higher ratios of H₂ to CO₂ would favor alcohols over corresponding carboxylic acids, or longer-chain acids and alcohols over shorter-chain acids and alcohols. Some organisms might even make alkanes instead of acids or alcohols. Therefore, the team hypothesized, it may be possible to isolate more ideal organisms for conversion of gases to fuels by increasing pressure and adjusting ratios of gases in the fermentation.

Isolates that produced alcohols included Enterococcus species (>97% homology with E. avium, E. faecium, E. faecalis), and Clostridium species, and unidentified strictly anaerobic gram-negative spiral rods. A strain of Enterococcus avium was examined in additional studies. When ethanol was initially included in the media at 6% concentration by volume, 3 to 4 times greater ethanol was produced from gases than when it was not included and acid production ceased. Alcohols represented greater than 90% of the measured (C₂–C₅) volatile fatty acids plus alcohols on a molar basis. Of the alcohol production, 83% was ethanol, 11% 1-propanol, 3% iso-propanol, and 3% 1-butanol.

Cultures selected from hydrocarbon enrichments produced alkanes including 1-hexane and other hydrocarbons of similar size (C₅ to C₈). Forty-six of fifty isolates grew in broth, and 41 produced medium-length (C₃–C₈) hydrocarbons from CO₂ and H₂. The 41 isolates that produced alkanes were identified by 16-S rRNA, which were >97% homologous with at least one of Actinomyces sp., Enterococcus faecium, E. Hirae, Eschericia coli, Clostridium glycolicum, Proteus sp., or Tissierella sp.

Previously there were no organisms known to produce medium-length hydrocarbons. … In the first attempt to isolate alkane-producing organisms using the second law of thermodynamics, 82% of isolated organisms produced alkanes from H₂ and CO₂. It is not yet known if the fuel production rates and the robustness of the organisms will allow for their use in commercial fuel production. However, the process described in this article proves the concept that it is possible to isolate organisms for biofuel or chemical production by applying the laws of thermodynamics.

Classic isolation procedures grew cultures of microorganisms on a certain feedstuff and then identified the compounds that individual isolates produced. Finding an organism that produces a desired product was a matter of chance. Growing cultures in high concentrations of the desired products (e.g. ethanol or alkanes) without consideration of the thermodynamics selects for degradation of the products rather than their production. The present approach enriches for organisms that produce certain types of products at a high concentration.

—Kohn and Kim

Funding was provided with a Grant from the Maryland Agricultural Experiment Station, University of Maryland.

Resources

• Richard A. Kohn, Seon-Woo Kim (2015) “Using the second law of thermodynamics for enrichment and isolation of microorganisms to produce fuel alcohols or hydrocarbons,” Journal of Theoretical Biology, Volume 382, Pages 356-362 doi: 10.1016/j.jtbi.2015.07.019

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